Model biological systems demonstrate the inducibility of pathways that strongly reduce cryoprotectant toxicity.

Cryoprotectant toxicity is a limiting factor for the cryopreservation of many living systems. We were moved to address this problem by the potential of organ vitrification to relieve the severe shortage of viable donor organs available for human transplantation. The M22 vitrification solution is presently the only solution that has enabled the vitrification and subsequent transplantation with survival of large mammalian organs, but its toxicity remains an obstacle to organ stockpiling for transplantation. We therefore undertook a series of exploratory studies to identify potential pretreatment interventions that might reduce the toxic effects of M22. Hormesis, in which a living system becomes more resistant to toxic stress after prior subtoxic exposure to a related stress, was investigated as a potential remedy for M22 toxicity in yeast, in the nematode worm C. elegans, and in mouse kidney slices. In yeast, heat shock pretreatment increased survival by 18-fold after exposure to formamide and by over 9-fold after exposure to M22 at 30 °C; at 0 °C and with two-step addition, treatment with 90% M22 resulted in 100% yeast survival. In nematodes, surveying a panel of pretreatment interventions revealed 3 that conferred nearly total protection from acute whole-worm M22-induced damage. One of these protective pretreatments (exposure to hydrogen peroxide) was applied to mouse kidney slices in vitro and was found to strongly protect nuclear and plasma membrane integrity in both cortical and medullary renal cells exposed to 75-100% M22 at room temperature for 40 min. These studies demonstrate for the first time that endogenous cellular defenses, conserved from yeast to mammals, can be marshalled to substantially ameliorate the toxic effects of one of the most toxic single cryoprotectants and the toxicity of the most concentrated vitrification solution so far described for whole organs.

A molecular sensor determines the ubiquitin substrate specificity of SARS-CoV-2 papain-like protease.

The SARS-CoV-2 papain-like protease (PLpro) is a target for antiviral drug development. It is essential for processing viral polyproteins for replication and functions in host immune evasion by cleaving ubiquitin (Ub) and ubiquitin-like protein (Ubl) conjugates. While highly conserved, SARS-CoV-2 and SARS-CoV PLpro have contrasting Ub/Ubl substrate preferences. Using a combination of structural analyses and functional assays, we identify a molecular sensor within the S1 Ub-binding site of PLpro that serves as a key determinant of substrate specificity. Variations within the S1 sensor specifically alter cleavage of Ub substrates but not of the Ubl interferon-stimulated gene 15 protein (ISG15). Significantly, a variant of concern associated with immune evasion carries a mutation in the S1 sensor that enhances PLpro activity on Ub substrates. Collectively, our data identify the S1 sensor region as a potential hotspot of variability that could alter host antiviral immune responses to newly emerging SARS-CoV-2 lineages.

Activity profiling and crystal structures of inhibitor-bound SARS-CoV-2 papain-like protease: A framework for anti-COVID-19 drug design.

Viral papain-like cysteine protease (PLpro, NSP3) is essential for SARS-CoV-2 replication and represents a promising target for the development of antiviral drugs. Here, we used a combinatorial substrate library and performed comprehensive activity profiling of SARS-CoV-2 PLpro. On the scaffold of the best hits from positional scanning, we designed optimal fluorogenic substrates and irreversible inhibitors with a high degree of selectivity for SARS PLpro. We determined crystal structures of two of these inhibitors in complex with SARS-CoV-2 PLpro that reveals their inhibitory mechanisms and provides a molecular basis for the observed substrate specificity profiles. Last, we demonstrate that SARS-CoV-2 PLpro harbors deISGylating activity similar to SARSCoV-1 PLpro but its ability to hydrolyze K48-linked Ub chains is diminished, which our sequence and structure analysis provides a basis for. Together, this work has revealed the molecular rules governing PLpro substrate specificity and provides a framework for development of inhibitors with potential therapeutic value or drug repurposing.

Activity profiling and structures of inhibitor-bound SARS-CoV-2-PLpro protease provides a framework for anti-COVID-19 drug design.

In December 2019, the first cases of a novel coronavirus infection causing COVID-19 were diagnosed in Wuhan, China. Viral Papain-Like cysteine protease (PLpro, NSP3) is essential for SARS-CoV-2 replication and represents a promising target for the development of antiviral drugs. Here, we used a combinatorial substrate library containing natural and a wide variety of nonproteinogenic amino acids and performed comprehensive activity profiling of SARS-CoV-2-PLpro. On the scaffold of best hits from positional scanning we designed optimal fluorogenic substrates and irreversible inhibitors with a high degree of selectivity for SARS PLpro variants versus other proteases. We determined crystal structures of two of these inhibitors (VIR250 and VIR251) in complex with SARS-CoV-2-PLpro which reveals their inhibitory mechanisms and provides a structural basis for the observed substrate specificity profiles. Lastly, we demonstrate that SARS-CoV-2-PLpro harbors deISGylating activities similar to SARS-CoV-1-PLpro but its ability to hydrolyze K48-linked Ub chains is diminished, which our sequence and structure analysis provides a basis for. Altogether this work has revealed the molecular rules governing PLpro substrate specificity and provides a framework for development of inhibitors with potential therapeutic value or drug repositioning.

The T-cell leukemia related rpl10-R98S mutant traps the 60S export adapter Nmd3 in the ribosomal P site in yeast.

Mutations in the ribosomal protein Rpl10 (uL16) can be drivers of T-cell acute lymphoblastic leukemia (T-ALL). We previously showed that these T-ALL mutations disrupt late cytoplasmic maturation of the 60S ribosomal subunit, blocking the release of the trans-acting factors Nmd3 and Tif6 in S. cerevisiae. Consequently, these mutant ribosomes do not efficiently pass the cytoplasmic quality control checkpoint and are blocked from engaging in translation. Here, we characterize suppressing mutations of the T-ALL-related rpl10-R98S mutant that bypass this block and show that the molecular defect of rpl10-R98S is a failure to release Nmd3 from the P site. Suppressing mutations were identified in Nmd3 and Tif6 that disrupted interactions between Nmd3 and the ribosome, or between Nmd3 and Tif6. Using an in vitro system with purified components, we found that Nmd3 inhibited Sdo1-stimulated Efl1 activity on mutant rpl10-R98S but not wild-type 60S subunits. Importantly, this inhibition was overcome in vitro by mutations in Nmd3 that suppressed rpl10-R98S in vivo. These results strongly support a model that Nmd3 must be dislodged from the P site to allow Sdo1 activation of Efl1, and define a failure in the removal of Nmd3 as the molecular defect of the T-ALL-associated rpl10-R98S mutation.

Nmd3 is a structural mimic of eIF5A, and activates the cpGTPase Lsg1 during 60S ribosome biogenesis.

During ribosome biogenesis in eukaryotes, nascent subunits are exported to the cytoplasm in a functionally inactive state. 60S subunits are activated through a series of cytoplasmic maturation events. The last known events in the cytoplasm are the release of Tif6 by Efl1 and Sdo1 and the release of the export adapter, Nmd3, by the GTPase Lsg1. Here, we have used cryo-electron microscopy to determine the structure of the 60S subunit bound by Nmd3, Lsg1, and Tif6. We find that a central domain of Nmd3 mimics the translation elongation factor eIF5A, inserting into the E site of the ribosome and pulling the L1 stalk into a closed position. Additional domains occupy the P site and extend toward the sarcin-ricin loop to interact with Tif6. Nmd3 and Lsg1 together embrace helix 69 of the B2a intersubunit bridge, inducing base flipping that we suggest may activate the GTPase activity of Lsg1.

Understanding Biases in Ribosome Profiling Experiments Reveals Signatures of Translation Dynamics in Yeast.

Ribosome profiling produces snapshots of the locations of actively translating ribosomes on messenger RNAs. These snapshots can be used to make inferences about translation dynamics. Recent ribosome profiling studies in yeast, however, have reached contradictory conclusions regarding the average translation rate of each codon. Some experiments have used cycloheximide (CHX) to stabilize ribosomes before measuring their positions, and these studies all counterintuitively report a weak negative correlation between the translation rate of a codon and the abundance of its cognate tRNA. In contrast, some experiments performed without CHX report strong positive correlations. To explain this contradiction, we identify unexpected patterns in ribosome density downstream of each type of codon in experiments that use CHX. These patterns are evidence that elongation continues to occur in the presence of CHX but with dramatically altered codon-specific elongation rates. The measured positions of ribosomes in these experiments therefore do not reflect the amounts of time ribosomes spend at each position in vivo. These results suggest that conclusions from experiments in yeast using CHX may need reexamination. In particular, we show that in all such experiments, codons decoded by less abundant tRNAs were in fact being translated more slowly before the addition of CHX disrupted these dynamics.

Bypass of the pre-60S ribosomal quality control as a pathway to oncogenesis.

Ribosomopathies are a class of diseases caused by mutations that affect the biosynthesis and/or functionality of the ribosome. Although they initially present as hypoproliferative disorders, such as anemia, patients have elevated risk of hyperproliferative disease (cancer) by midlife. Here, this paradox is explored using the rpL10-R98S (uL16-R98S) mutant yeast model of the most commonly identified ribosomal mutation in acute lymphoblastic T-cell leukemia. This mutation causes a late-stage 60S subunit maturation failure that targets mutant ribosomes for degradation. The resulting deficit in ribosomes causes the hypoproliferative phenotype. This 60S subunit shortage, in turn, exerts pressure on cells to select for suppressors of the ribosome biogenesis defect, allowing them to reestablish normal levels of ribosome production and cell proliferation. However, suppression at this step releases structurally and functionally defective ribosomes into the translationally active pool, and the translational fidelity defects of these mutants culminate in destabilization of selected mRNAs and shortened telomeres. We suggest that in exchange for resolving their short-term ribosome deficits through compensatory trans-acting suppressors, cells are penalized in the long term by changes in gene expression that ultimately undermine cellular homeostasis.

Eukaryotic rpL10 drives ribosomal rotation.

Ribosomes transit between two conformational states, non-rotated and rotated, through the elongation cycle. Here, we present evidence that an internal loop in the essential yeast ribosomal protein rpL10 is a central controller of this process. Mutations in this loop promote opposing effects on the natural equilibrium between these two extreme conformational states. rRNA chemical modification analyses reveals allosteric interactions involved in coordinating intersubunit rotation originating from rpL10 in the core of the large subunit (LSU) through both subunits, linking all the functional centers of the ribosome. Mutations promoting rotational disequilibria showed catalytic, biochemical and translational fidelity defects. An rpL3 mutation promoting opposing structural and biochemical effects, suppressed an rpL10 mutant, re-establishing rotational equilibrium. The rpL10 loop is also involved in Sdo1p recruitment, suggesting that rotational status is important for ensuring late-stage maturation of the LSU, supporting a model in which pre-60S subunits undergo a 'test drive' before final maturation.

Regulation of ribosome biogenesis by nucleostemin 3 promotes local and systemic growth in Drosophila.

Nucleostemin 3 (NS3) is an evolutionarily conserved protein with profound roles in cell growth and viability. Here we analyze cell-autonomous and non-cell-autonomous growth control roles of NS3 in Drosophila and demonstrate its GTPase activity using genetic and biochemical assays. Two null alleles of ns3, and RNAi, demonstrate the necessity of NS3 for cell autonomous growth. A hypomorphic allele highlights the hypersensitivity of neurons to lowered NS3 function. We propose that NS3 is the functional ortholog of yeast and human Lsg1, which promotes release of the nuclear export adapter from the large ribosomal subunit. Release of the adapter and its recycling to the nucleus are essential for sustained production of ribosomes. The ribosome biogenesis role of NS3 is essential for proper rates of translation in all tissues and is necessary for functions of growth-promoting neurons.

Exome sequencing identifies mutation in CNOT3 and ribosomal genes RPL5 and RPL10 in T-cell acute lymphoblastic leukemia.

T-cell acute lymphoblastic leukemia (T-ALL) is caused by the cooperation of multiple oncogenic lesions. We used exome sequencing on 67 T-ALLs to gain insight into the mutational spectrum in these leukemias. We detected protein-altering mutations in 508 genes, with an average of 8.2 mutations in pediatric and 21.0 mutations in adult T-ALL. Using stringent filtering, we predict seven new oncogenic driver genes in T-ALL. We identify CNOT3 as a tumor suppressor mutated in 7 of 89 (7.9%) adult T-ALLs, and its knockdown causes tumors in a sensitized Drosophila melanogaster model. In addition, we identify mutations affecting the ribosomal proteins RPL5 and RPL10 in 12 of 122 (9.8%) pediatric T-ALLs, with recurrent alterations of Arg98 in RPL10. Yeast and lymphoid cells expressing the RPL10 Arg98Ser mutant showed a ribosome biogenesis defect. Our data provide insights into the mutational landscape of pediatric versus adult T-ALL and identify the ribosome as a potential oncogenic factor.